Probiotic bacteria have been investigated in the prevention and treatment of various diseases and allergies. The current study was undertaken to determine the effect of eight probiotic Lactobacillus species against bacterial human skin pathogens using several techniques. Antimicrobial activity of lactobacilli against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Propionibacterium acnes was evaluated using lactobacilli broth cultures (BCB) and cell free supernatant (CFS). Antimicrobial activity was significantly greater with BCB compared with CFS especially for Lactobacillus plantarum and Lactobacillus acidophilus. Lactobacilli and pathogen aggregation, biofilm formation and adhesion to keratin were assessed. L. casei and L. plantarum were selected for further study as they showed the greatest co-aggregation (18.02 ± 1.34% with L. casei and 14.92 ± 1.45% with L. plantarum) with the pathogens (16.63 ± 1.65% with S. aureus 3761 and 14.58 ± 1.68% with P. aeruginosa) and prevention of biofilm formation by the pathogens. The antimicrobial activity of human beta defensin-2 (hBD-2) alone or with L. plantarum against pathogens was assessed. The results with hBD-2 showed that hBD-2 (10 μg / ml for 5 h) and L. plantarum together were significantly more inhibitory against S. aureus than hBD-2 alone. The presence of NaCl reduced the effectiveness of hBD-2 alone and with L. plantarum. In the presence of L. plantarum, inactivation of mprF and dlt genes led to increased binding of hBD-2 by the bacterial cell wall, and then inhibition growth of bacterial cell wall. Studies investigated the effect of exposure of methicillin resistant Staphylococcus aureus (MRSA) to the supernatant of L. plantarum the susceptibility of MRSA to β-lactams. MRSA became sensitive to β-lactams when treated with culture supernatant of L. plantarum. Gene expression studies demonstrated that the mecR1-mecI-mecA-PBP2 signalling pathway was impeded by exposure to culture supernatant of L. plantarum and β-lactams. The studies reported here demonstrate a possible alternative approach to dealing with skin pathogens, which may have clinical implications especially with regard to MRSA infections, and continued research is advised.